Modified polypeptide with attenuated activity of citrate synthase and method for producing L-amino acid using the same

ABSTRACT

The present disclosure relates to a modified polypeptide with attenuated activity of citrate synthase and a method for producing an aspartate-derived L-amino acid using the modified polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/470,030 filed Jun. 14, 2019, now pending, which is a U.S.national phase application of PCT/KR2019/001697, filed Feb. 12, 2019,which claims priority to KR Application No. 10-2018-0017400, filed Feb.13, 2018. U.S. application Ser. No. 16/470,030 is herein incorporated byreference in its entity.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing200187_444D1_SEQUENCE_LISTING.xml (Size: 85,744 bytes; and Date ofCreation: Oct. 4, 2022) is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present disclosure relates to a modified polypeptide with attenuatedactivity of citrate synthase and a method for producing L-amino acidusing the modified polypeptide.

BACKGROUND ART

A microorganism of the genus Corynebacterium, specificallyCorynebacterium glutamicum, is a Gram-positive microorganism that iswidely used in the production of L-amino acid and other usefulmaterials. For the production of the L-amino acid and other usefulmaterials, various studies are underway to develop microorganisms withhigh-efficiency production and technologies for fermentation processes.For example, target material specific approaches (e.g., increasing theexpression of genes encoding the enzymes involved in L-lysinebiosynthesis or removing genes unnecessary for biosynthesis) are mainlyused (KR Patent No. 10-0838038).

Meanwhile, among L-amino acids, L-lysine, L-threonine, L-methionine,L-isoleucine, and L-glycine are aspartate-derived amino acids, and thebiosynthesis level of oxaloacetate (i.e., a precursor of aspartate) canaffect the biosynthesis levels of these L-amino acids.

Citrate synthase (CS) is an enzyme that produces citrate by catalyzingthe condensation of acetyl-CoA and oxaloacetate produced duringglycolysis of a microorganism, and it is also an important enzyme fordetermining carbon-flow into the TCA pathway.

The phenotypic changes in L-lysine-producing strains due to the deletionof gltA gene encoding citrate synthase were reported previously in aliterature (Ooyen et al., Biotechnol. Bioeng., 109(8): 2070-2081, 2012).However, these strains with the deletion of gltA gene have disadvantagesin that not only their growth is inhibited but also their sugarconsumption rates are significantly reduced thus resulting in low lysineproduction per unit time. Accordingly, there is still a need for studiesin which an effective increase in L-amino acid productivity and thegrowth of the strains can be both considered.

DISCLOSURE Technical Problem

The present inventors have confirmed that when a novel modifiedpolypeptide in which citrate synthase activity is attenuated to acertain level is used, the amount of L-amino acid production can beincreased without delay in the growth rate of the strain, therebycompleting the present disclosure.

Technical Solution

An object of the present disclosure is to provide a modified polypeptidewith citrate synthase activity, in which the 241^(st) amino acid in theamino acid sequence of SEQ ID NO: 1, asparagine, is substituted withanother amino acid.

Another object of the present disclosure is to provide a polynucleotideencoding the modified polypeptide.

Still another object of the present disclosure is to provide amicroorganism of the genus Corynebacterium producing anaspartate-derived L-amino acid, comprising the modified polypeptide.

Still another object of the present disclosure is to provide a methodfor producing an L-amino acid, which includes culturing themicroorganism of the genus Corynebacterium in a medium; and recoveringan L-amino acid from the cultured microorganism or medium.

Advantageous Effect

When the novel modified polypeptide of the present disclosure withattenuated citrate synthase activity is used, the amount ofaspartate-derived L-amino acid production can be further improvedwithout delaying the growth rate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the growth curve of a strain in which deletion andmodification of gltA gene are introduced.

BEST MODE

The present disclosure is described in detail as follows. Meanwhile,respective descriptions and embodiments disclosed in the presentdisclosure may also be applied to other descriptions and embodiments.That is, all combinations of various elements disclosed in the presentdisclosure fall within the scope of the present disclosure. Further, thescope of the present disclosure is not limited by the specificdescription below.

To achieve the above objects, an aspect of the present disclosureprovides a modified polypeptide having citrate synthase activity, inwhich the modified polypeptide includes at least one modification in theamino acid of SEQ ID NO: 1 and the at least one modification includessubstitution of the 241^(st) amino acid in the amino acid sequence ofSEQ ID NO: 1 (i.e., asparagine) with another amino acid.

Specifically, the modified polypeptide may be described as a modifiedpolypeptide having citrate synthase activity, in which the 241^(st)amino acid in the amino acid sequence of SEQ ID NO: 1 (i.e., asparagine)is substituted with another amino acid.

In the present disclosure, the SEQ ID NO: 1 refers to an amino acidsequence having the activity of citrate synthase, and specifically, aprotein sequence having the activity of citrate synthase encoded by gltAgene. The amino acid sequence of SEQ ID NO: 1 may be obtained from NCBIGenBank, which is a public database. For example, the amino acidsequence of SEQ ID NO: 1 may be derived from Corynebacterium glutamicum,but the amino acid sequence is not limited thereto, and may include anysequence having the same activity as that of the above amino acidsequence without limitation. Further, the amino acid sequence mayinclude the amino acid sequence of SEQ ID NO: 1 or any amino acidsequence having 80% or more homology or identity to the amino acidsequence of SEQ ID NO: 1, but the amino acid sequence is not limitedthereto. Specifically, the amino acid sequence may include the aminoacid sequence of SEQ ID NO: 1 and any amino acid sequence having atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology to the aminoacid sequence of SEQ ID NO: 1. Further, it is apparent that any proteinhaving an amino acid sequence, in which part of the amino acid sequenceis deleted, modified, substituted, or added, may also be used in thepresent disclosure as long as the protein has such a homology oridentity in an amino acid sequence to that of the above protein andexhibits an effect corresponding to that of the above protein.

That is, in the present disclosure, although it is described as “proteinor polypeptide having an amino acid sequence of a particular SEQ ID NO”or “protein or polypeptide consisting of an amino acid sequence of aparticular SEQ ID NO”, it is apparent that any protein which hasbiological activity substantially the same as or equivalent to thepolypeptide consisting of the amino acid sequence of the correspondingSEQ ID NO may be used in the present disclosure, even if the amino acidsequence may have deletion, modification, substitution, or addition inpart of the sequence. For example, it is apparent that the “polypeptideconsisting of an amino acid sequence of SEQ ID NO: 1” can belong to the“polypeptide consisting of an amino acid sequence of SEQ ID NO: 1”.Additionally, in a case where the polypeptide has activity the same asor equivalent to the modified polypeptide of the present disclosure, amutation that can occur due to a meaningless sequence addition upstreamor downstream of the amino acid sequence of the corresponding SEQ ID NO,a naturally occurring mutation, or a silent mutation therein is notexcluded, in addition to a modification on the 241^(st) amino acid or amodification corresponding thereto, and it is apparent that in caseswhere the polypeptide has such a sequence addition or mutation, theresulting peptides can also belong to the scope of the presentdisclosure.

As used herein, the term “homology” or “identity” represents relevancebetween two given amino acid sequences or nucleotide sequences and maybe expressed as percentage. These two terms “homology” and “identity”are often used interchangeably with each other.

The sequence homology or identity of conserved polynucleotide orpolypeptide sequences may be determined by standard alignment algorithmsand may be used with default gap penalty established by the programbeing used. Substantially homologous or identical sequences aregenerally expected to hybridize under moderate or high stringentconditions, along the entire length or at least about 50%, about 60%,about 70%, about 80%, or about 90% of the entire length of the targetpolynucleotides or polypeptides. With respect to the hybridization,polynucleotides that contain degenerate codons instead of the codons inthe hybridizing polypeptides are also considered.

Whether any two polynucleotide or polypeptide sequences have a homologyor identity may be determined using a known computer algorithm, such asthe “FASTA” program using default parameters as described by Pearson etal. (Proc. Natl. Acad. Sci. USA 85: 2444, (1988)). Alternatively, thehomology or identity may be determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), whichis performed in the Needleman program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277) (preferably, version 5.0.0 or versionsthereafter) (GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] etal., J Molec Bio 215]: 403 (1990); Guide to Huge Computers, Martin J.Bishop, [ED.], Academic Press, San Diego, 1994, and [CARILLOETA/.](1988) SIAM J Applied Math 48: 1073). For example, the homology,similarity, or identity may be determined using BLAST or ClustalW of theNational Center for Biotechnology Information.

The homology, similarity, or identity between polynucleotides orpolypeptides may be determined by comparing sequence information using,for example, the GAP computer program (e.g., Needleman et al. (1970), JMol Biol. 48: 443) as disclosed in the literature (Smith and Waterman,Adv. Appl. Math (1981) 2:482). In summary, the GAP program defineshomology, similarity, or identity as the value obtained by dividing thenumber of similarly aligned symbols (i.e., nucleotides or amino acids)by the total number of the symbols in the shorter of the two sequences.Default parameters for the GAP program may include (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) and the weighted comparison matrix of Gribskov et al.(1986), Nucl. Acids Res. 14: 6745, as disclosed in the literature(Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap (or a gap opening penalty of 10 and a gap extensionpenalty of 0.5); and (3) no penalty for end gaps.

Additionally, whether any two polynucleotide or polypeptide sequenceshave a homology, similarity, or identity may be determined by comparingthese sequences via Southern hybridization experiments, and theappropriate hybridization conditions to be defined may be determined bya method known to those skilled in the art (e.g., J. Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press, Cold Spring Harbor, N.Y., 1989; F. M. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork).

As used herein, the term “modified polypeptide” refers to a polypeptide,in which one or more amino acids differ from those of the recitedsequence in conservative substitution and/or modification, but thefunctions or properties of the polypeptide are maintained. The modifiedpolypeptide differs from those sequences which are identified bysubstitution, deletion, or addition of several amino acids. Such amodified polypeptide can generally be identified by modifying one of thepolypeptide sequences and evaluating the properties of the modifiedpolypeptide. That is, the ability of a modified polypeptide may beincreased, unchanged, or decreased relative to that of the nativeprotein. Such a modified polypeptide can generally be identified bymodifying one of the polypeptide sequences and evaluating the reactivityof the modified polypeptide. Additionally, a partially modifiedpolypeptide may include a modified polypeptide in which one or moreparts therein (e.g., a N-terminal leader sequence, a transmembranedomain, etc.) are removed. Other modified polypeptides may include thosepolypeptides in which a part therein is removed from the N- and/orC-terminus of each mature protein. In the present disclosure, the term“modified” may be used interchangeably with terms, such as modification,modified protein, modified polypeptide, mutant, mutein, divergent,variant, etc., and the term is not limited as long as it is used as ameaning of modification.

As used herein, the term “conservative substitution” refers tosubstitution of an amino acid with another amino acid having similarstructural and/or chemical properties. The variant(modified polypeptide)may have, for example, one or more conservative substitutions whilemaintaining one or more biological activities. This amino acidsubstitution may be generally performed based on similarity in polarity,electric charge, solubility, hydrophobicity, hydrophilicity, and/oramphipathic nature of amino acid residues. For example, among theelectrically charged amino acids, positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include glutamic acid and aspartic acid; and among theuncharged amino acids, nonpolar amino acids include glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, andproline; polar or hydrophilic amino acids include serine, threonine,cysteine, tyrosine, asparagine, and glutamine; and among the polar aminoacids, aromatic amino acids include phenylalanine, tryptophan, andtyrosine.

In addition, variants (modified polypeptide) may include deletion oraddition of amino acids having a minimal effect on the characteristicsand secondary structure of a polypeptide. For example, a polypeptide maybe conjugated to a signal (or leader) sequence of a protein N-terminusthat is involved in the transfer of proteins co-translationally orpost-translationally. Additionally, the polypeptide may also beconjugated to another sequence or linker to be identified, purified, orsynthesized.

The modified polypeptide of the present disclosure may be a modifiedpolypeptide with attenuated citrate synthase activity compared to thatof the amino acid sequence of SEQ ID NO: 1, in which the modifiedpolypeptide includes at least one modification in the amino acid of SEQID NO: 1, and the at least one modification includes substitution of the241^(st) amino acid in the amino acid sequence of SEQ ID NO: 1 (i.e.,asparagine) with another amino acid. The modified polypeptide may bedescribed as a modified polypeptide with attenuated citrate synthaseactivity compared to that of the amino acid sequence of SEQ ID NO: 1, inwhich the 241^(st) amino acid in the amino acid sequence of SEQ ID NO: 1is substituted with another amino acid.

The “substitution with another amino acid” is not limited as long as theamino acid substituted differs from the amino acid before substitution.That is, when the 241^(st) amino acid in the amino acid sequence of SEQID NO: 1 substituted with another amino acid, the another amino acid isnot limited as long as the another amino acid is an amino acid otherthan asparagine.

The modified polypeptide of the present disclosure may be one which hasreduced or attenuated activity of citrate synthase compared to thepolypeptide before modification, native wild-type polypeptide, orunmodified polypeptide, but the modified polypeptide is not limitedthereto.

Specifically, the modified polypeptide of the present disclosure may beone, in which the 241^(st) amino acid in the amino acid sequence of SEQID NO: 1 (i.e., asparagine) is substituted with glycine, alanine,arginine, aspartate, cysteine, glutamate, glutamine, histidine, proline,serine, tyrosine, isoleucine, leucine, lysine, tryptophan, valine,methionine, phenylalanine, or threonine. More specifically, the modifiedpolypeptide may be one, in which the 241^(st) amino acid in the aminoacid sequence of SEQ ID NO: 1 (i.e., asparagine) is substituted with anamino acid other than lysine, but the substitution is not limitedthereto. Alternatively, the modified polypeptide may be one, in whichthe 241^(st) amino acid in the amino acid sequence of SEQ ID NO: 1(i.e., asparagine) is substituted with an amino acid other than anacidic or basic amino acid, or substituted with an amino acid having anuncharged amino acid, but the substitution is not limited thereto.Alternatively, the modified polypeptide may be one, in which the241^(st) amino acid in the amino acid sequence of SEQ ID NO: 1 (i.e.,asparagine) is substituted with a nonpolar amino acid or hydrophilicamino acid, and specifically with an aromatic amino acid (e.g.,phenylalanine, tryptophan, and tyrosine) or a hydrophilic amino acid(e.g., serine, threonine, tyrosine, cysteine, asparagine, andglutamine), but the modified polypeptide is not limited thereto. Morespecifically, the modified polypeptide may be one with attenuatedcitrate synthase activity, in which the 241^(st) amino acid in the aminoacid sequence of SEQ ID NO: 1 (i.e., asparagine) is substituted withthreonine, serine, or tyrosine, but the modified polypeptide is notlimited thereto. Even more specifically, the modified polypeptide may beone with attenuated citrate synthase activity, in which the 241^(st)amino acid in the amino acid sequence of SEQ ID NO: 1 (i.e., asparagine)is substituted with threonine, but the modified polypeptide is notlimited thereto.

Such a modified polypeptide has citrate synthase activity which isattenuated compared to that of the polypeptide having the amino acidsequence of SEQ ID NO: 1. It is apparent that the modified polypeptide,in which the 241^(st) amino acid in the amino acid sequence of SEQ IDNO: 1 is substituted with another amino acid, includes modifiedpolypeptides in which any amino acid corresponding to the 241^(st) aminoacid is substituted with another amino acid.

Specifically, among the modified polypeptides, the modified polypeptidein which the 241^(st) amino acid in the amino acid sequence of SEQ IDNO: 1 (i.e., asparagine) is substituted with another amino acid may beone which consists of SEQ ID NOS: 3, 59, and 61, and more specifically,the modified polypeptide in which the 241^(st) amino acid in the aminoacid sequence of SEQ ID NO: 1 (i.e., asparagine) is substituted withthreonine, serine, or tyrosine may be one which consists of each of SEQID NOS: 3, 59, and 61, but the modified polypeptide is not limitedthereto. Additionally, the modified polypeptide may include the aminoacid sequences of SEQ ID NOS: 3, 59, and 61, or amino acid sequenceswhich have at least 80% of a homology to each of the amino acidsequences of SEQ ID NOS: 3, 59, and 61, but the modified polypeptide isnot limited thereto. Specifically, the modified polypeptide of thepresent disclosure may include the amino acid sequences of SEQ ID NOS:3, 59, and 61, or amino acid sequences which have at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% of a homology to each of the amino acid sequences of SEQ ID NOS: 3,59, and 61. Additionally, it is apparent that any protein having anamino acid sequence with deletion, modification, substitution, oraddition in part of the amino acid sequence, in addition to the 241^(st)amino acid of the amino acid sequence, can also be included in the scopeof the present disclosure, as long as the amino acid sequence has thehomology described above and has an effect corresponding to that of theprotein.

As used herein, the term “citrate synthase (CS)” refers to an enzymethat produces citrate by catalyzing the condensation of acetyl-CoA andoxaloacetate produced during glycolysis of a microorganism, and it is animportant enzyme that determines carbon-flow into the TCA pathway.Specifically, citrate synthase acts as a factor to regulate the rate inthe first step of the TCA cycle as an enzyme for synthesizing citrate.In addition, the citrate synthase catalyzes the condensation reaction ofthe two-carbon acetate residue from acetyl-CoA and a molecule of4-carbon oxaloacetate to form the 6-carbon acetate. In the presentdisclosure, the citrate synthase may be used interchangeably with“enzyme for synthesizing citrate” or “CS”.

Another aspect of the present disclosure provides a polynucleotideencoding the modified polypeptide.

As used herein, the term “polynucleotide”, which is a polymer ofnucleotides composed of nucleotide monomers connected in a lengthy chainby a covalently bond, is a DNA or RNA strand having at least a certainlength, and more specifically, a polynucleotide fragment encoding themodified polypeptide.

The polynucleotide encoding the modified polypeptide of the presentdisclosure may include without limitation any polynucleotide sequenceencoding a modified polypeptide with attenuated citrate synthaseactivity of the present disclosure. In the present disclosure, the geneencoding the amino acid sequence of the citrate synthase polypeptide maybe the gltA gene, specifically a gene derived from Corynebacteriumglutamicum, but the gene is not limited thereto.

The polynucleotide of the present disclosure may undergo variousmodifications in the coding region without changing the amino acidsequence of the polynucleotide, due to codon degeneracy or inconsideration of the codons preferred in an organism in which thepolynucleotide is to be expressed. Specifically, any polynucleotidesequence encoding the modified polypeptide, in which the 241^(st) aminoacid in the amino acid sequence of SEQ ID NO: 1 is substituted withanother amino acid, may be included without limitation. For example, thepolynucleotide of the present disclosure may be those which consist ofthe amino acid sequences of SEQ ID NOS: 3, 59, and 61, respectively, orpolynucleotide sequences encoding polypeptides having a sequencehomology to these polypeptides, but the polynucleotide is not limitedthereto. More specifically, the polynucleotide of the present disclosuremay one which consists of each polynucleotide sequence of SEQ ID NOS: 4,60, and 62, but the polynucleotide of the present disclosure is notlimited thereto.

Additionally, a probe that may be prepared from a known gene sequence,for example, any sequence which can hybridize with a sequencecomplementary to all or part of the polynucleotide sequence understringent conditions to encode a protein having the activity of themodified polypeptide in which the 241^(st) amino acid in the amino acidsequence of SEQ ID NO: 1 is substituted with another amino acid, may beincluded without limitation.

The “stringent conditions” refers to conditions under which specifichybridization between polynucleotides is allowed. Such conditions arespecifically described in the literature (e.g., J. Sambrook et al.,supra). The stringent conditions may include conditions under whichgenes having a high homology, for example, 40% or higher homology,specifically 90% or higher homology, more specifically 95% or higherhomology, much more specifically 97% or higher homology, still much morespecifically 99% or higher homology are hybridized with each other andgenes having a homology lower than the above homologies are nothybridized with each other, or ordinary washing conditions of Southernhybridization (i.e., washing once, specifically, twice or three times ata salt concentration and a temperature corresponding to 60° C., 1×SSC,0.1% SDS, specifically, 60° C., 0.1×SSC, 0.1% SDS, and more specifically68° C., 0.1×SSC, 0.1% SDS), but the stringent conditions are not limitedthereto and may be appropriately adjusted by those skilled in the art.

Hybridization requires that two polynucleotides contain complementarysequences, although mismatches between bases are possible depending onthe stringency of the hybridization. The term “complementary” is used todescribe the relationship between nucleotide bases that can hybridizewith each other. For example, with respect to DNA, adenosine iscomplementary to thymine and cytosine is complementary to guanine.Therefore, the present disclosure may include isolated nucleotidefragments complementary to the entire sequence as well as polynucleotidesequences substantially similar thereto.

Specifically, the polynucleotides having a homology may be detectedusing the hybridization conditions including a hybridization step at aT_(m) value of 55° C. under the above-described conditions. Further, theT_(m) value may be 60° C., 63° C., or 65° C., but the T_(m) value is notlimited thereto and may be appropriately adjusted by those skilled inthe art depending on the purpose thereof.

The appropriate stringency for hybridizing polynucleotides depends onthe length of the polynucleotides and the degree of complementation, andthese variables are well known in the art (see Sambrook et al., supra,9.50-9.51, 11.7-11.8).

Still another aspect of the present disclosure provides a microorganismcomprising the modified polypeptide. Specifically, the presentdisclosure provides a microorganism of the genus Corynebacteriumproducing L-amino acid, comprising the modified polypeptide. Morespecifically, the present disclosure provides a microorganism of thegenus Corynebacterium producing aspartate-derived L-amino acid,comprising the modified polypeptide. For example, the microorganism tobe provided may be one which is transformed with a vector containing apolynucleotide encoding the modified polypeptide, but the microorganismis not limited thereto.

The microorganisms comprising the modified polypeptide of the presentdisclosure have an improved ability to produce L-amino acid compared tothe microorganism comprising a wild-type polypeptide, without inhibitionof growth or sugar consumption rate of the microorganism. Therefore,L-amino acid can be obtained in high yield from these microorganisms.Specifically, it may be interpreted that the microorganism comprisingthe modified polypeptide can establish a suitable balance between thecarbon flow into the TCA pathway and the supply amount of oxaloacetateused as a precursor of the biosynthesis of L-amino acid by controllingthe activity of citrate synthase, and as a result, the microorganismcomprising the modified polypeptide can increase the amount of L-aminoacid production, but the interpretation is not limited thereto.

As used herein, the term “L-amino acid” refers to an organic compoundcontaining amine and carboxyl functional group, and specifically, anamino acid in α-amino acid form or a L stereoisomer form (L-form). TheL-amino acid may be asparagine, glycine, alanine, arginine, aspartate,cysteine, glutamic acid, glutamine, histidine, proline, serine,tyrosine, isoleucine, leucine, lysine, tryptophan, valine, methionine,phenylalanine, or threonine. Additionally, the L-amino acid may beL-homoserine (an α-amino acid) or a derivative thereof as a precursor ofL-amino acid, but the L-amino acid is not limited thereto. TheL-homoserine derivative may be, for example, one selected from the groupconsisting of O-acetylhomoserine, O-succinylhomoserine, andO-phosphohomoserine, but the L-homoserine derivative is not limitedthereto.

As used herein, the term “aspartate” refers to an α-amino acid which isused in the biosynthesis of proteins and may be used interchangeablywith aspartic acid. Generally, aspartate is produced from oxaloacetate,which is a precursor of aspartate, and may be converted to L-lysine,L-methionine, L-homoserine or a derivative thereof, L-threonine,L-isoleucine, etc. in vivo.

As used herein, the term “aspartate-derived L-amino acid” refers to amaterial which can be biosynthesized using aspartate as a precursor, andthe aspartate-derived L-amino acid is not limited as long as the L-aminoacid can be produced via biosynthesis using aspartate as a precursor.The aspartate-derived L-amino acid may include not onlyaspartate-derived L-amino acid but also a derivative thereof. Forexample, the L-amino acid and derivative thereof may be L-lysine,L-threonine, L-methionine, L-glycine, homoserine or a derivative thereof(O-acetylhomoserine, O-succinylhomoserine, and O-phosphohomoserine),L-isoleucine, and/or cadaverine, but the L-amino acid and a derivativethereof are not limited thereto. Specifically, the L-amino acid andderivative thereof may be L-lysine, L-threonine, L-methionine,homoserine or a derivative thereof, and/or L-isoleucine, and morespecifically, the L-amino acid and derivative thereof may be L-lysine,L-threonine and/or L-isoleucine, but the L-amino acid and derivativethereof are not limited thereto.

As used herein, the term “vector” refers to a DNA product including anucleotide sequence of a polynucleotide encoding a target protein, whichis operably linked to a suitable control sequence to express the targetprotein in a suitable host. The control sequence includes a promotercapable of initiating transcription, an arbitrary operator sequence forcontrolling transcription, a sequence encoding an appropriate mRNAribosome-binding site, and sequences for controlling the termination oftranscription and translation. Once transformed into a suitable hostcell, the vector may replicate or function independently of the hostgenome or may be integrated into the genome itself.

The vector used in the present disclosure is not particularly limited aslong as it can replicate in a host cell, and any vector known in the artmay be used. Examples of the vector conventionally used may includenatural or recombinant plasmids, cosmids, viruses, and bacteriophages.For example, as a phage vector or cosmid vector, pWE15, M13, MBL3, MBL4,IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., may be used; andas a plasmid vector, those based on pBR, pUC, pBluescriptII, pGEM, pTZ,pCL, pET, etc., may be used. Specifically, pDZ, pACYC177, pACYC184, pCL,pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc., may be used.

The vector that can be used in the present disclosure is notparticularly limited, and any known expression vector may be used. Inaddition, a polynucleotide encoding a target protein may be insertedinto the chromosome using a vector for intracellular chromosomalinsertion. The insertion of the polynucleotide into the chromosome maybe performed by any method known in the art (e.g., homologousrecombination), but the method is not limited thereto. The vector mayfurther include a selection marker to confirm a successful geneinsertion into the chromosome. The selection marker is for screening thecells transformed with the vector, that is, for determining whether thetarget polynucleotide molecule has been inserted. Markers that provideselectable phenotypes (e.g., drug resistance, auxotrophy, resistance tocell toxic agents, or expression of surface proteins) may be used. Underthe circumstances treated with a selective agent, only the cellsexpressing the selection marker can survive or express other phenotypictraits, and thus the transformed cells can be selected.

As used herein, the term “transformation” refers to the introduction ofa vector including a polynucleotide encoding a target protein into ahost cell so that the protein encoded by the polynucleotide can beexpressed in a host cell. As long as the transformed polynucleotide canbe expressed in the host cell, it does not matter whether thetransformed polynucleotide is integrated into the chromosome the hostcell and located therein or located extrachromosomally. Further, thepolynucleotide includes DNA and RNA encoding the target protein. Thepolynucleotide may be introduced in any form, as long as it can beintroduced into the host cell and expressed therein. For example, thepolynucleotide may be introduced into the host cell in the form of anexpression cassette, which is a gene construct including all elementsrequired for its autonomous expression. The expression cassette mayinclude a promoter operably linked to the polynucleotide, atranscription terminator, a ribosome binding site, or a translationterminator. The expression cassette may be in the form of aself-replicable expression vector. In addition, the polynucleotide maybe introduced into the host cell as it is and operably linked tosequences required for expression in the host cell, but the introductionof the polynucleotide into the cell is not limited thereto. Thetransformation method includes any method of introducing apolynucleotide into a cell, and may be performed by selecting a suitablestandard technique known in the art, depending on the host cell. Forexample, the method may include electroporation, calcium phosphate(Ca(H₂PO₄)₂, CaHPO₄, or Ca₃(PO₄)₂) precipitation, calcium chloride(CaCl₂)) precipitation, microinjection, a polyethyleneglycol (PEG)method, a DEAE-dextran method, a cationic liposome method, a lithiumacetate-DMSO method, etc., but the method is not limited thereto.

Additionally, as used herein, the term “operable linkage” means that thepolynucleotide sequence is functionally linked to a promoter sequencethat initiates and mediates transcription of the polynucleotide encodingthe target protein of the present disclosure. The operable linkage maybe prepared using a gene recombinant technique known in the art, andsite-specific DNA cleavage and linkage may be prepared using enzymes forcleavage and ligation known in the art, etc., but the preparation of theoperable linkage is not limited thereto.

As used herein, the term “microorganism comprising a modifiedpolypeptide” refers to a host cell or microorganism which includes apolynucleotide encoding a modified polypeptide, or a host cell ormicroorganism which is transformed with a vector including apolynucleotide encoding a modified polypeptide and is thus able toexpress the modified polypeptide therein. The host cell or microorganismmay be of native wild-type or one in which natural or artificial geneticmodification has occurred. Specifically, the microorganism of thepresent disclosure may be one having citrate synthase activity, in whichthe 241^(st) amino acid, asparagine, is substituted with another aminoacid, thus expressing a modified polypeptide, but the microorganism isnot limited thereto. Additionally, the microorganism including amodified polypeptide may be a microorganism that produces an L-aminoacid. Specifically, the microorganism comprising a modified polypeptidemay be a microorganism with an improved ability of L-amino acidproduction compared to its native type or unmodified parent strain, butthe microorganism is not limited thereto. Additionally, themicroorganism comprising a modified polypeptide may be a microorganismthat produces an aspartate-derived L-amino acid. Specifically, themicroorganism including a modified polypeptide may be a microorganismwith an improved ability of aspartate-derived L-amino acid productioncompared to its native type or unmodified parent strain, but themicroorganism is not limited thereto.

Examples of the microorganism may include a microorganism strain of thegenus Escherichia, Serratia, Erwinia, Enterobacteria, Salmonella,Streptomyces, Pseudomonas, Brevibacterium, Corynebacterium, etc., andspecifically a microorganism of the genus Corynebacterium.

For example, the microorganism of the genus Corynebacterium may beCorynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacteriumlactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes,Corynebacterium efficiens, etc., but the microorganism of the genusCorynebacterium is not necessarily limited thereto. More specifically,the microorganism of the genus Corynebacterium may be Corynebacteriumglutamicum, but the microorganism is not limited thereto.

In a specific embodiment, the microorganism may be a microorganism thatproduces L-lysine, in which the modification of gltA is introduced intoa microorganism of the genus Corynebacterium where the activities of theproteins encoded by three kinds of modified pyc, hom, and lysC genes areincreased and thereby the ability to produce L-lysine is increased.

Additionally, the microorganism may be a microorganism that producesL-threonine and L-isoleucine, in which a modification is introduced intoa gene encoding homoserine dehydrogenase that produces homoserine (i.e.,a common intermediate in the biosynthesis pathways for L-threonine andL-isoleucine) thereby enhancing the activity of the gene, but themicroorganism is not limited thereto. In particular, the microorganismmay be a microorganism that produces L-isoleucine, in which a furthermodification is introduced into a gene encoding L-threonine dehydratasethereby enhancing the activity of the gene, but the microorganism is notlimited thereto. Accordingly, for the purpose of the present disclosure,the microorganism that produces an L-amino acid may be a microorganismin which the modified polypeptide is further added and thereby theability of producing the target L-amino acid is increased.

Another aspect of the present disclosure provides a method for producingan L-amino acid, which includes culturing the microorganism in a medium;and recovering an L-amino acid from the cultured microorganism ormedium. Specifically, the L-amino acid may be an aspartate-derivedL-amino acid.

The method may be easily determined by those skilled in the art underthe optimized culture conditions and enzyme activity conditions.Specifically, the microorganism may be cultured by a known batchculture, continuous culture, fed-batch culture, etc., but the culturemethod is not particularly limited thereto. In particular, the cultureconditions are not particularly limited, but the pH (e.g., pH 5 to pH 9,specifically pH 6 to pH 8, and most specifically pH 6.8) may beappropriately adjusted using a basic compound (e.g., sodium hydroxide,potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoricacid or sulfuric acid). An aerobic condition may be maintained by addingoxygen or an oxygen-containing gas mixture to the culture. The culturetemperature may be maintained at 20° C. to 45° C., and specifically at25° C. to 40° C., and the cultivation may be performed for about 10 to160 hours, but the culture conditions not limited thereto. The L-aminoacid produced by the cultivation may be secreted into the medium or mayremain within the cells.

Additionally, in the culture medium, carbon sources, such as sugars andcarbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose,molasses, starch, and cellulose), oils and fats (e.g., soybean oil,sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g.,palmitic acid, stearic acid, and linoleic acid), alcohols (e.g.,glycerol and ethanol), and organic acids (e.g., acetic acid), may beused alone or in combination, but the carbon sources are not limitedthereto; nitrogen sources, such as nitrogen-containing organic compounds(e.g., peptone, yeast extract, meat juice, malt extract, corn steepliquor, soybean flour, and urea), or inorganic compounds (e.g., ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate), may be used alone or in combination, but the nitrogensources are not limited thereto; and potassium sources, such aspotassium dihydrogen phosphate, dipotassium hydrogen phosphate, orsodium-containing salts corresponding thereto, may be used alone or incombination, but the potassium sources are not limited thereto.Additionally, other essential growth-stimulating materials includingmetal salts (e.g., magnesium sulfate or iron sulfate), amino acids, andvitamins may be contained in the medium.

In the method of recovering the L-amino acid produced in the cultivationstep of the present disclosure, it is possible to collect the targetamino acid from the culture using an appropriate method known in the artaccording to the cultivation method. For example, centrifugation,filtration, anion exchange chromatography, crystallization, HPLC, etc.may be used, and the desired L-amino acid may be recovered from themedium or microorganism using an appropriate method known in the art.

Further, the recovering step may include a purification process. Thepurification process may be performed using an appropriate method knownin the art. Therefore, the L-amino acid being recovered may be in apurified form or a microorganism fermentation liquid containing theL-amino acid.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present disclosure will be described in detail throughexemplary embodiments. However, these exemplary embodiments are forillustrative purposes only and are not intended to limit the scope ofthe present disclosure.

Example 1: Preparation of Vector Library for Introducing Modification inORF of gltA Gene

For the purpose of discovering modified strains in which the expressionlevel or activity of gltA gene of Corynebacterium glutamicum isattenuated, a library was prepared by the following method.

First, 0 to 4.5 modifications were introduced per 1 kb of a DNA fragment(1,814 bp) including the gltA gene (1,314 bp) using the GenemorphIIRandom Mutagenesis Kit (Stratagene). Error-prone PCR was performed usingthe chromosome of Corynebacterium glutamicum ATCC13032 (WT) as atemplate along with primers (SEQ ID NOS: 5 and 6) (Table 1).Specifically, the reaction solution containing the chromosome of the WTstrain (500 ng), primers 5 and 6 (125 ng each), Mutazyme II reactionbuffer (1×), dNTP mix (40 mM), and Mutazyme II DNA polymerase (2.5 U)was subjected to denaturation at 94° C. for 2 minutes followed by 25cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1minute, and polymerization at 72° C. for 3 minutes, and thenpolymerization at 72° C. for 10 minutes.

The amplified gene fragments were ligated to a pCRII vector using theTOPO TA Cloning Kit (Invitrogen), transformed into E. coli DH5α, and thetransformed E. coli DH5α was plated on a solid LB medium containingkanamycin (25 mg/L). 20 Kinds of transformed colonies were selected andthe plasmids obtained therefrom were subjected to sequence analysis. Asa result, it was confirmed that modifications were introduced into sitesdifferent from each other at a frequency of 0.5 mutations/kb. Finally,about 10,000 E. coli transformed colonies were collected and theplasmids were extracted therefrom and named as pTOPO-gltA(mt) library.

TABLE 1 Primer Sequence (5′ → 3′) Primer (SEQ ID NO: 5)ATGTTTGAAAGGGATATCGTG Primer (SEQ ID NO: 6) TTAGCGCTCCTCGCGAGGAAC

Example 2: Preparation of gltA-Deleted Strains and Screening ofgltA-Modified Strains Based on Growth Rate

To prepare a strain of wild-type Corynebacterium glutamicum ATCC13032 inwhich the gltA gene is deleted, the pDZ-ΔgltA vector in which the gltAgene is deleted was prepared as follows. Specifically, the pDZ-ΔgltAvector (KR Patent No. 10-0924065) was prepared such that the DNAfragments (600 bp each) located at 5′ and 3′ of the gltA gene wereligated to the pDZ-ΔgltA vector. Primers (SEQ ID NOS: 7 and 8), in whicha recognition site of the restriction enzyme (XbaI) was inserted at the5′ fragment and the 3′ fragment, respectively, based on the nucleotidesequence of the reported gltA gene (SEQ ID NO: 2), and primers (SEQ IDNOS: 9 and 10), each located 600 bp apart from the primers (SEQ ID NOS:7 and 8), were synthesized (Table 2). The 5′-end fragment was preparedby PCR using the chromosome of the Corynebacterium glutamicum ATCC13032as a template along with primers (SEQ ID NOS: 7 and 9). Likewise, thegene fragment located at the 3′ end of the gltA gene was prepared by PCRusing the primers (SEQ ID NOS: 8 and 10). PCR was performed as follows:denaturation at 94° C. for 2 minutes; 30 cycles of denaturation at 94°C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at72° C. for 40 seconds; and polymerization at 72° C. for 10 minutes.

Meanwhile, the DNA fragments amplified by PCR as described above wereligated to the pDZ vector, which was cleaved with a restriction enzyme(XbaI) and then heat-treated at 65° C. for 20 minutes, transformed intoE. coli DH5α, and the transformed E. coli DH5α was plated on a solid LBmedium containing kanamycin (25 mg/L). Colonies transformed with thevector, in which the target gene was inserted thereinto by PCR using theprimers (SEQ ID NOS: 7 and 8), were selected and the plasmid wasobtained from the colonies using a conventional plasmid extractionmethod and named as pDZ-ΔgltA.

TABLE 2 Primer Sequence (5′ → 3′) Primer (SEQ IDCGGGGATCCTCTAGACGATGAAAAACGCCC NO: 7) Primer (SEQ IDCAGGTCGACTCTAGACTGCACGTGGATCGT NO: 8) Primer (SEQ IDACTGGGACTATTTGTTCGGAAAAA NO: 9) Primer (SEQ ID CGAACAAATAGTCCCAGTTCAACGNO: 10)

The prepared pDZ-ΔgltA vector was transformed into Corynebacteriumglutamicum ATCC13032 by an electric pulse method (Van der Rest et al.,Appl. Microbiol. Biotechnol. 52:541-545, 1999), and the strain in whichthe gltA gene is deleted was prepared by homologous recombination. ThegltA gene-deleted strain was named as Corynebacterium glutamicumWT::ΔgltA.

Additionally, the pTOPO-gltA(mt) library was transformed by an electricpulse method using the WT:ΔgltA strain. The transformed strain wasplated on a composite plate medium containing kanamycin (25 mg/L) andabout 500 colonies were obtained therefrom. The obtained colonies wereinoculated into a 96-well plate in which a seed culture medium (200μL/well) was contained, and the strain was cultured at 32° C. at a rateof 1,000 rpm for about 9 hours.

<Composite Plate Medium (pH 7.0)>

Glucose (10 g), Peptone (10 g), Beef Extract (5 g), Yeast Extract (5 g),Brain Heart Infusion (18.5 g), NaCl (2.5 g), Urea (2 g), Sorbitol (91g), Agar (20 g) (based on 1 L of distilled water)

<Seed Culture Medium (pH 7.0)>

Glucose (20 g), Peptone (10 g), Yeast Extract (5 g), Urea (1.5 g),KH₂HPO₄ (4 g), K₂HPO₄ (8 g), MgSO₄.7H₂O (0.5 g), Biotin (100 μg),Thiamine.HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg),Nicotinamide (2,000 μg) (based on 1 L of distilled water)

The cell growth during culture was monitored using theUV-spectrophotometer micro-reader (Shimazu) (FIG. 1 ). WT and WT::ΔgltAstrains were used as control groups. Three kinds of strains, in whichthe cell mass was smaller but the cell growth rate was maintained at ahigher rate compared to that of the wild-type (WT) strain, were selectedand named as WT::gltA(mt)-1 to 3. The remaining 497 strains showed asimilar or increased cell mass or a reduced growth rate compared tothose of WT and WT::ΔgltA strains (control groups).

Example 3: Confirmation of Nucleotide Sequences of Three gltA-ModifiedStrains

To confirm the nucleotide sequences of the gltA gene of the threeselected strains (i.e., WT::gltA(mt)-1 to 3), the DNA fragmentsincluding the gltA gene in the chromosome were amplified using theprimers (SEQ ID NOS: 5 and 6) specified in Example 1. PCR was performedas follows: denaturation at 94° C. for 2 minutes; 30 cycles ofdenaturation at 94° C. for 1 minute, annealing at 56° C. for 1 minute,and polymerization at 72° C. for 40 seconds; and polymerization at 72°C. for 10 minutes.

The nucleotide sequences of the amplified genes were analyzed, and as aresult, it was confirmed that these nucleotide sequences commonly showedthe introduction of 1 to 2 modifications into the nucleotide sequencelocated 721 bp to 723 bp downstream of the gltA gene ORF initiationcodon. That is, it was confirmed that the WT::gltA(mt)-1 to 3 strainswere modified strains of citrate synthase (CS) in which the 721^(st) tothe 723^(rd) nucleotide sequences are changed from the original ‘AAC’ to‘ACC’ or ‘ACT’ (i.e., the change of the 241^(st) amino acid from theN-terminus of the gltA gene from asparagine to threonine).

Example 4: Preparation of Various Strains in which the 241^(st) AminoAcid of gltA Gene (i.e., Asparagine) is Substituted with Another AminoAcid

An attempt was made to substitute the 241^(st) amino acid in the aminoacid sequence of SEQ ID NO: 1 (i.e., asparagine) (possessed by thewild-type strain) with an amino acid other than asparagine.

To introduce 19 kinds of modifications of heterogeneous nucleotidesubstitution including the N241T, which is the modification confirmed inExample 3, each recombinant vector was prepared as follows.

First, primers (SEQ ID NOS: 11 and 12), in which a recognition site ofthe restriction enzyme (XbaI) was inserted into the 5′ fragment and the3′ fragment, respectively, about 600 bp apart either downstream orupstream from the positions of the 721^(st) to the 723^(rd) nucleotidesequences of the gltA gene, were synthesized using the genomic DNAextracted from the WT strain as a template. To introduce the 19 kinds ofheterogeneous nucleotide-substituted modifications, primers (SEQ ID NOS:13 to 48) for substituting the 721^(st) to the 723^(rd) nucleotidesequences of the gltA gene were synthesized (Table 3).

Specifically, the pDZ-gltA(N241A) plasmid was prepared in such a formthat the DNA fragments (600 bp each) located at the 5′ and 3′ ends ofthe gltA gene were ligated to the pDZ vector (Korea Patent No.2009-0094433). The 5′ end gene fragment of the gltA gene was prepared byPCR using the chromosome of WT strain as a template along with primers(SEQ ID NOS: 11 and 13). PCR was performed as follows: denaturation at94° C. for 2 minutes; 30 cycles of denaturation at 94° C. for 1 minute,annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40seconds; and polymerization at 72° C. for 10 minutes. Likewise, the 3′end gene fragment of the gltA gene was prepared by PCR using primers(SEQ ID NOS: 12 and 14). The amplified DNA fragments were purified usingthe PCR Purification kit (Quiagen) and used as insertion DNA fragmentsfor the preparation of vectors.

Meanwhile, the insertion DNA fragments amplified by PCR and the pDZvector, which was cleaved with a restriction enzyme (XbaI) and thenheat-treated at 65° C. for 20 minutes, were ligated using the InfusionCloning Kit and then transformed into E. coli DH5α. The strain wasplated on a solid LB medium containing kanamycin (25 mg/L). Thetransformed colonies in which the target gene was inserted into thevector by PCR using the primers (SEQ ID NOS: 11 and 12) were selected,and the plasmid was obtained using a conventionally known plasmidextraction method and named as pDZ-gltA(N241A).

Likewise, plasmids were prepared as follows: the pDZ-gltA(N241V) usingprimers (SEQ ID NOS: 11 and 15 and SEQ ID NOS: 12 and 16); thepDZ-gltA(N241Q) using primers (SEQ ID NOS: 11 and 17 and SEQ ID NOS: 12and 18); the pDZ-gltA(N241H) using primers (SEQ ID NOS: 11 and 19 andSEQ ID NOS: 12 and 20); the pDZ-gltA(N241R) using primers (SEQ ID NOS:11 and 21 and SEQ ID NOS: 12 and 22); the pDZ-gltA(N241P) using primers(SEQ ID NOS: 11 and 23 and SEQ ID NOS: 12 and 24); the pDZ-gltA(N241L)using primers (SEQ ID NOS: 11 and 25 and SEQ ID NOS: 12 and 26); thepDZ-gltA(N241Y) using primers (SEQ ID NOS: 11 and 27 and SEQ ID NOS: 12and 28); the pDZ-gltA(N241S) using primers (SEQ ID NOS: 11 and 29 andSEQ ID NOS: 12 and 30); the pDZ-gltA(N241K) using primers (SEQ ID NOS:11 and 31 and SEQ ID NOS: 12 and 32); the pDZ-gltA(N241M) using primers(SEQ ID NOS: 11 and 33 and SEQ ID NOS: 12 and 34); the pDZ-gltA(N241I)using primers (SEQ ID NOS: 11 and 35 and SEQ ID NOS: 12 and 36); thepDZ-gltA(N241E) using primers (SEQ ID NOS: 11 and 37 and SEQ ID NOS: 12and 38); the pDZ-gltA(N241D) using primers (SEQ ID NOS: 11 and 39 andSEQ ID NOS: 12 and 40); the pDZ-gltA(N241G) using primers (SEQ ID NOS:11 and 41 and SEQ ID NOS: 12 and 42); the pDZ-gltA(N241W) using primers(SEQ ID NOS: 11 and 43 and SEQ ID NOS: 12 and 44); the pDZ-gltA(N241C)using primers (SEQ ID NOS: 11 and 45 and SEQ ID NOS: 12 and 46); thepDZ-gltA(N241F) using primers (SEQ ID NOS: 11 and 47 and SEQ ID NOS: 12and 48); and the pDZ-gltA(N241T) using primers (SEQ ID NOS: 11 and 49and SEQ ID NOS: 12 and 50).

TABLE 3 Primer Sequence (5′ → 3′) Primer (SEQ ID NO: 11)CGGGGATCCTCTAGAAGATGCTGTCTGAGACTGGA Primer (SEQ ID NO: 12)CAGGTCGACTCTAGACGCTAAATTTAGCGCTCCTC Primer (SEQ ID NO: 13)GGAGGTGGAGCATGCCTGCTCGTGGTCAGC Primer (SEQ ID NO: 14)GACCACGAGCAGGCATGCTCCACCTCCACC Primer (SEQ ID NO: 15)GGAGGTGGAGCAGACCTGCTCGTGGTCAGC Primer (SEQ ID NO: 16)GACCACGAGCAGGTCTGCTCCACCTCCACC Primer (SEQ ID NO: 17)GGAGGTGGAGCACTGCTGCTCGTGGTCAGC Primer (SEQ ID NO: 18)GACCACGAGCAGCAGTGCTCCACCTCCACC Primer (SEQ ID NO: 19)GGAGGTGGAGCAGTGCTGCTCGTGGTCAGC Primer (SEQ ID NO: 20)GACCACGAGCAGCACTGCTCCACCTCCACC Primer (SEQ ID NO: 21)GGAGGTGGAGCAGCGCTGCTCGTGGTCAGC Primer (SEQ ID NO: 22)GACCACGAGCAGCGCTGCTCCACCTCCACC Primer (SEQ ID NO: 23)GGAGGTGGAGCATGGCTGCTCGTGGTCAGC Primer (SEQ ID NO: 24)GACCACGAGCAGCCATGCTCCACCTCCACC Primer (SEQ ID NO: 25)GGAGGTGGAGCACAGCTGCTCGTGGTCAGC Primer (SEQ ID NO: 26)GACCACGAGCAGCTGTGCTCCACCTCCACC Primer (SEQ ID NO: 27)GGAGGTGGAGCAGTACTGCTCGTGGTCAGC Primer (SEQ ID NO: 28)GACCACGAGCAGTACTGCTCCACCTCCACC Primer (SEQ ID NO: 29)GGAGGTGGAGCAGGACTGCTCGTGGTCAGC Primer (SEQ ID NO: 30)GACCACGAGCAGTCCTGCTCCACCTCCACC Primer (SEQ ID NO: 31)GGAGGTGGAGCACTTCTGCTCGTGGTCAGC Primer (SEQ ID NO: 32)GACCACGAGCAGAAGTGCTCCACCTCCACC Primer (SEQ ID NO: 33)GGAGGTGGAGCACATCTGCTCGTGGTCAGC Primer (SEQ ID NO: 34)GACCACGAGCAGATGTGCTCCACCTCCACC Primer (SEQ ID NO: 35)GGAGGTGGAGCAGATCTGCTCGTGGTCAGC Primer (SEQ ID NO: 36)GACCACGAGCAGATCTGCTCCACCTCCACC Primer (SEQ ID NO: 37)GGAGGTGGAGCATTCCTGCTCGTGGTCAGC Primer (SEQ ID NO: 38)GACCACGAGCAGGAATGCTCCACCTCCACC Primer (SEQ ID NO: 39)GGAGGTGGAGCAGTCCTGCTCGTGGTCAGC Primer (SEQ ID NO: 40)GACCACGAGCAGGACTGCTCCACCTCCACC Primer (SEQ ID NO: 41)GGAGGTGGAGCAGCCCTGCTCGTGGTCAGC Primer (SEQ ID NO: 42)GACCACGAGCAGGGCTGCTCCACCTCCACC Primer (SEQ ID NO: 43)GGAGGTGGAGCAGCACTGCTCGTGGTCAGC Primer (SEQ ID NO: 44)GACCACGAGCAGTGGTGCTCCACCTCCACC Primer (SEQ ID NO: 45)GGAGGTGGAGCAGCACTGCTCGTGGTCAGC Primer (SEQ ID NO: 46)GACCACGAGCAGTGCTGCTCCACCTCCACC Primer (SEQ ID NO: 47)GGAGGTGGAGCAGAACTGCTCGTGGTCAGC Primer (SEQ ID NO: 48)GACCACGAGCAGTTCTGCTCCACCTCCACC Primer (SEQ ID NO: 49)GGAGGTGGAGCAGGTCTGCTCGTGGTCAGC Primer (SEQ ID NO: 50)GACCACGAGCAGACCTGCTCCACCTCCACC

Each of the prepared vectors was transformed into the lysine-producingCorynebacterium glutamicum KCCM11016P strain (KR Patent No. 10-0159812)by the electric pulse method. The 19 strains in which a modification ofheterogeneous nucleotide substitution was introduced to the gltA gene ofeach strain were named as follows: KCCM11016P::gltA(N241A),KCCM11016P::gltA(N241V), KCCM11016P::gltA(N241Q),KCCM11016P::gltA(N241H), KCCM11016P::gltA(N241R),KCCM11016P::gltA(N241P), KCCM11016P::gltA(N241L),KCCM11016P::gltA(N241Y), KCCM11016P::gltA(N241S),KCCM11016P::gltA(N241K), KCCM11016P::gltA(N241M),KCCM11016P::gltA(N241I), KCCM11016P::gltA(N241E),KCCM11016P::gltA(N241D), KCCM11016P::gltA(N241G),KCCM11016P::gltA(N241W), KCCM11016P::gltA(N241C),KCCM11016P::gltA(N241F), and KCCM11016P::gltA(N241T).

Example 5: Analysis of Lysine Productivity and Measurement of CitrateSynthase (CS) Activity of gltA-Modified Strains

The citrate synthase (CS) activity of the strains prepared in Example 4was measured by the previously reported method (Ooyen et al.,Biotechnol. Bioeng., 109(8): 2070-2081, 2012). The gltA gene of theKCCM11016P strain was deleted by the method used in Example 1 and theresulting strain was named as KCCM11016P::ΔgltA. While using KCCM11016Pand KCCM11016P::ΔgltA strains as the control groups, the selected 19kinds of strains were cultured as described below, and the sugarconsumption rate, production yield of lysine, concentration of glutamicacid (GA) (i.e., a representative by-product in the cultured medium),and CS enzyme activity were measured.

First, each of the strains was inoculated into a 250 mL corner-baffleflask containing 25 mL of a seed culture medium and cultured in ashaking incubator (200 rpm) at 30° C. for 20 hours. Then, each of the250 mL corner-baffle flasks containing 24 mL of an L-lysine productionmedium was inoculated with 1 mL of a seed culture broth and cultured ina shaking incubator (200 rpm) at 32° C. for 72 hours. The compositionsof the seed culture medium and the production medium are shown below.After the completion of the culture, the concentrations of L-lysine andglutamic acid in each culture were measured by HPLC (Waters 2478).

<Seed Culture Medium (pH 7.0)>

Glucose (20 g), Peptone (10 g), Yeast Extract (5 g), Urea (1.5 g),KH₂PO₄ (4 g), K₂HPO₄ (8 g), MgSO₄. 7H₂O (0.5 g), Biotin (100 μg),Thiamine.HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg),Nicotinamide (2,000 μg) (based on 1 L of distilled water)

<Production Medium (pH 7.0)>

Glucose (100 g), (NH₄)₂SO₄ 40 g, (40 g), Soybean Protein (2.5 g), CornSteep Solids (5 g), Urea (3 g), KH₂PO₄ (1 g), MgSO₄.7H₂O (0.5 g), Biotin(100 μg), Thiamine.HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg),Nicotinamide (3,000 μg), and CaCO3 (30 g) (based on 1 L of distilledwater)

To measure the CS enzyme activity, the cells were recovered bycentrifugation, washed twice with 100 mM Tris-HCl buffer (pH 7.2, 3 mML-cysteine, 10 mM MgCl₂), and the resultant was finally suspended in 2mL of the same buffer. The cell suspension was physically disrupted for10 minutes in a conventional glass bead vortexing method and thesupernatant was recovered by two rounds of centrifugation (13,000 rpm,4° C., 30 minutes) and used as a crude extract for the measurement of CSenzyme activity. To measure the CS enzyme activity, the crude extractwas added to a reaction liquid for enzyme activity measurement (50 mMTris, 200 mM potassium glutamate, pH 7.5, 0.1 mM 5,50-Dithiobis(2-nitrobenzoic acid, DTNB), 0.2 mM oxaloacetate, 0.15 mM acetyl-CoA)and reacted at 30° C. The CS activity was defined in terms of a rationby measuring the absorbance of DTNB, decomposed per minute relative tothe parent strain, at 412 nm. The results of lysine-producing ability,sugar consumption rates, compositions of culture broth, and CS enzymeactivity are shown in Table 4 below.

TABLE 4 Measurement of lysine-producing ability, compositions of culturebroth, and CS enzyme activity (%) GA sugar CS Activity LYS YieldConcentration Consumption Strain (%) (%) (mg/L) Rate (g/hr) KCCM11016P100 43.4 436 4.53 KCCM11016P::ΔgltA 2 49.0 13 1.31KCCM11016P::gltA(N241A) 36 46.2 430 3.56 KCCM11016P::gltA(N241V) 61 44.8428 4.08 KCCM11016P::gltA(N241Q) 91 43.9 386 4.21KCCM11016P::gltA(N241H) 57 44.0 431 4.33 KCCM11016P::gltA(N241R) 86 43.5432 4.68 KCCM11016P::gltA(N241P) 71 43.9 411 4.66KCCM11016P::gltA(N241L) 79 44.7 429 4.51 KCCM11016P::gltA(N241Y) 35 46.9373 4.59 KCCM11016P::gltA(N241S) 36 46.8 391 4.48KCCM11016P::gltA(N241K) 61 44.1 409 4.19 KCCM11016P::gltA(N241M) 52 44.0412 3.89 KCCM11016P::gltA(N2411) 41 46.5 422 3.65KCCM11016P::gltA(N241E) 51 43.8 401 3.90 KCCM11016P::gltA(N241D) 40 46.1399 3.51 KCCM11016P::gltA(241G) 71 44.9 418 4.12 KCCM11016P::gltA(N241W)45 46.2 308 3.54 KCCM11016P::gltA(241C) 46 46.6 310 3.69KCCM11016P::gltA(N241F) 48 45.7 386 4.09 KCCM11016P::gltA(241T) 31 48.6351 4.51

In the case of a strain where the gltA gene is deleted, the lysine yieldof the strain showed an increase of about 5.5% p compared to that of itsparent strain, but the strain was unable to consume sugar until thelater stage of the cultivation. That is, in the case where the gltA geneis deleted and thus the strain has almost no CS activity, the growth ofthe strain is inhibited thus making the industrial application of thestrain difficult. It was confirmed that in all cases where the strainincluded a modified polypeptide in which the 241^(st) amino acid in theamino acid sequence of SEQ ID NO: 1 was substituted with a differentamino acid, the CS activity was attenuated while the growth of thestrain was maintained at an industrially applicable level. Additionally,as the CS activity of the strain was attenuated, the lysine yield of thestrain tended to increase by about 3% p to 5% p compared to that of itsparent strain. In particular, in the cases of three modified strains(i.e., N241S, N241Y, and N241T) among the modified strains in which theCS activity was attenuated to a 30% to 60% level, these strains showedan increase in the lysine yield by about 3% p to 5% p compared to thatof their parent strain while showing similar levels in the sugarconsumption rate. Additionally, it was confirmed that the strains wherethe lysine yield was increased compared to that of their parent strainshowed a decrease in the amount of glutamic acid (GA) in the culturebroth. That is, it was interpreted that the introduction of amodification of the present disclosure into these strains has an effectof improving the lysine yield of these strains while reducing theby-products of these strains.

These results show that the amount of lysine production can be increasedvia an appropriate balance between the carbon-flow into the TCA pathwayand the amount of oxaloacetate (i.e., a precursor of lysinebiosynthesis) supply. In particular, considering that the amount ofglutamic acid, which is normally produced as a by-product in a largeamount during lysine cultivation, was reduced, it was confirmed that theattenuation of the gltA gene activity inhibits the carbon-flow into theTCA pathway and thereby induces the carbon-flow into the direction oflysine biosynthesis thus being significantly effective in increasing thelysine productivity.

Among the strains prepared above, the KCCM11016P::gltA(N241T) strain wasdeposited on Nov. 20, 2017, to the Korean Collection for Korean CultureCenter of Microorganisms (KCCM), an international depositary authorityunder the Budapest Treaty, and assigned Accession Number KCCM12154P.

Example 6: Analysis of Lysine-Producing Ability of SelectedgltA-Modified Strains

The modifications of three gltA-modified strains selected in Example 5were introduced into L-lysine-producing Corynebacterium glutamicumstrains (i.e., KCCM10770P (KR Patent No. 10-0924065) and KCCM11347P (KRPatent No. 10-0073610)). These three strains were selected based on thecriteria that they have reduced CS activity, have a sugar consumptionrate similar to that of their parent strain, and an increased lysineyield compared to that of their parent strain. The three kinds ofvectors of Example 4 (i.e., pDZ-gltA(N241S), pDZ-gltA(N241Y), andpDZ-gltA(N241T)) were introduced into the two Corynebacterium glutamicumstrains (i.e., KCCM10770P and KCCM11347P) by the electric pulse methodto prepare six strains (i.e., KCCM10770P::gltA(N241S),KCCM10770P::gltA(N241Y), KCCM10770P::gltA(N241T), KCCM11347P::gltA (N241S), KCCM11347P::gltA(N241Y), and KCCM11347P::gltA(N241T)). The twocontrol group strains (i.e., KCCM10770P and KCCM11347P) and the sixstrains with modifications of nucleotide substitution in the gltA genewere cultured in the same manner as in Example 5, and thelysine-producing ability, sugar consumption rate, and composition of theculture liquid of these strains were analyzed.

After culturing these strains for a certain period of time, the lysineproducing ability, sugar consumption rate and the composition of culturebroth were analyzed. The results are shown in Table 5 below.

TABLE 5 Analysis of lysine producing ability, sugar consumption rate andthe composition of culture broth, of gltA-modified strains GA Sugar LYSYield Concentration Consumption Strain (%) (mg/L) Rate (g/hr) KCCM10770P42.6 406 4.27 KCCM10770P::gltA(N241S) 46.4 350 4.08KCCM10770P::gltA(N241Y) 45.9 368 4.20 KCCM10770P::gltA(N241T) 48.0 2404.04 KCCM11347P 38.3 440 5.99 KCCM11347P::gltA(N241S) 41.7 386 5.85KCCM11347P::gltA(N241Y) 42.1 402 5.96 KCCM11347P::gltA(N241T) 43.5 3165.84

As shown in the results of Table 5 above, in the cases of twolysine-producing strains (i.e., KCCM10770P and KCCM11347P) where amodification that the 241^(st) amino acid of the gltA sequence wassubstituted with another amino acid, all of the strains showed anincreased lysine production yield, a decreased yield of by-products, anda similar sugar consumption rate compared to that of their parentstrain. It was confirmed that among the three kinds of modifications,the strain having the modification (N241T) (i.e., a modification wherethe 241^(st) amino acid (asparagine) was substituted with threonine)showed the highest increase in lysine yield while showing a similarlevel or slightly increased level of sugar consumption rate compared tothat of their parent strain. Additionally, it was confirmed that theN241T modification showed the highest level of decrease in glutamic acidyield. From these results, it was confirmed that the attenuation of thegltA gene activity resulted in the decrease into the TCA pathway therebycausing a decrease in the amount of glutamic acid in the culture broth,as confirmed in the results of Example 6.

Example 7: Preparation and Analysis of Lysine-Producing Ability of CJ3PStrain in which gltA-Modification (N241T) is Introduced

To confirm whether other L-lysine-producing Corynebacterium glutamicumstrains also have the same effect described above, a strain in which agltA(N241T) modification was introduced was prepared using theCorynebacterium glutamicum CJ3P (Binder et al. Genome Biology, 2012,13:R40) that is provided with the L-lysine-producing ability by theintroduction of three kinds of modifications [i.e., pyc(P458S),hom(V59A), and lysC(T311I)] to the wild-type strain in the same manneras in Example 6. The thus prepared strain was named as CJ3::gltA(N241T).The control groups (i.e., CJ3P and CJ3::gltA(N241T) strains) werecultured in the same manner as in Example 5, and the lysine producingability, sugar consumption rate and the composition of culture broth areanalyzed and the results are shown in Table 6 below.

TABLE 6 Analysis of lysine producing ability, sugar consumption rate andthe composition of culture broth of CJ3P-derived gltA-modified strainsLYS Yield GA Concentration Sugar Consumption Strain (%) (mg/L) Rate(g/hr) CJ3P 9.2 2689 5.86 CJ3P::gltA(N241T) 13.5 1983 5.51

As a result of the analysis of lysine producing ability, sugarconsumption rate and the composition of culture broth, it was confirmedthat the strain where the modification of gltA(N241T)) was introducedshowed an increase of lysine yield and a decrease in the concentrationof glutamic acid while maintaining the sugar consumption rate at asimilar level.

Example 8: Preparation of Threonine Strain in which gltA-Modification(N241T) is Introduced and Analysis of Threonine-Producing Ability

To explicitly confirm the changes in the L-threonine-producing abilityby the introduction of the gltA(N241T) modification, the gene encodinghomoserine dehydrogenase that produces homoserine (i.e., a commonintermediate in the biosynthesis pathways of L-threonine andL-isoleucine) was overexpressed by the modification. Specifically, astrain, in which a known hom(G378E) modification (R. Winkels, S. et al.,Appl. Microbiol. Biotechnol. 45, 612-620, 1996) was introduced into theCJ3P::gltA(N241T) strain used in Example 7, was prepared. Additionally,a strain, in which only the hom(G378E) modification was introduced tothe CJ3P, was prepared as the control group. Recombinant vectors for theintroduction of modifications were prepared by the method describedbelow.

To prepare a vector for the introduction of hom(G378E), first, primers(SEQ ID NO: 51 and 52), in which a recognition site of the restrictionenzyme (XbaI) was inserted into the 5′-end fragment and the 3′-endfragment, respectively, about 600 bp apart either downstream or upstreamfrom the positions of the 1,131^(st) to the 1,134^(th) nucleotidesequences of the hom gene, were synthesized using the genomic DNAextracted from the WT strain as a template. Primers (SEQ ID NO: 53 and54) for substituting the nucleotide sequence of the hom gene (Table 7).The pDZ-hom(G378E) plasmid was prepared in a form where the DNAfragments (600 bp each) located at each of the 5′ and 3′ ends wasconnected to the pDZ vector (Korea Patent No. 2009-0094433). The 5′-endfragment of the hom gene was prepared by PCR using the chromosome of theWT strain as a template along with primers (SEQ ID NO: 51 and 53). PCRwas performed as follows: denaturation at 94° C. for 2 minutes; 30cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1minute, and polymerization at 72° C. for 40 seconds; and polymerizationat 72° C. for 10 minutes. Likewise, 3′-end fragment of the hom gene wasprepared by PCR using primers (SEQ ID NO: 52 and 54). The amplified DNAfragment was purified using the PCR Purification kit (Quiagen) and usedas the insertion DNA fragment for vector preparation. Meanwhile, the pDZvector, which was treated with the restriction enzyme XbaI and heattreated at 65° C. for 20 minutes, was ligated to the insertion DNAfragment amplified by PCR using the Infusion Cloning Kit, and theligated product was transformed into E. coli DH5α and plated on a solidLB medium containing kanamycin (25 mg/L). The colonies, transformed witha vector in which the target gene obtained by PCR using primers (SEQ IDNO: 51 and 52) was inserted, were selected and the plasmid was obtainedby a conventionally known plasmid extraction method, and thereby avector for the introduction of a modification of nucleotide substitutionof hom(G378E) on the chromosome (i.e., pDZ-hom(G378E)) was prepared.

TABLE 7 Primer Sequence (5′ → 3′) Primer (SEQ ID NO:TCCTCTAGACTGGTCGCCTGATGTTCTAC 51) Primer (SEQ ID NO:GACTCTAGATTAGTCCCTTTCGAGGCGGA 52) Primer (SEQ ID NO:GCCAAAACCTCCACGCGATC 53) Primer (SEQ ID NO: ATCGCGTGGAGGTTTTGGCT 54)

Strains in which a nucleotide modification is introduced in the hom genein the CJ3P and CJ3P::gltA(N241T) strains by the pDZ-hom(G378E) vectorusing the method same as in Example 6 (i.e., CJ3P::hom(G378E) andCJ3P::gltA(N241T)-hom(G378E)) were obtained. The obtained two kinds ofstrains were cultured using the method same as in Example 5, and theconcentration of threonine, sugar consumption rate and composition ofthe culture broth were analyzed. The results are shown in Table 8 below.

TABLE 8 Concentration of threonine, sugar consumption rate, andcomposition of culture broth Thr GA Sugar Concentration ConcentrationConsumption Strain (g/L) (mg/L) Rate (g/hr) CJ3P::hom(G378E) 2.8 27695.36 CJ3P::(241T)- 6.1 1891 5.17 hom(G378E)

As a result of the analysis of the concentration of threonine, sugarconsumption rate and composition of the culture broth, it was confirmedthat the threonine concentration increased at a similar level of sugarconsumption rate while the glutamic acid concentration decreased, in thestrain where the modification of gltA(N241T) was introduced.

Example 9: Preparation of Isoleucine Strain where gltA-Modification(N241T) is Introduced and Analysis of Isoleucine-Producing Ability

To confirm the effect of the introduction of the gltA(N241T)modification on the L-isoleucine-producing ability, the gene encodingL-threonine dehydratase was also overexpressed by the previouslyreported modification. Specifically, a strain, in which a knownilvA(V323A) modification (S. Morbach et al., Appl. Enviro. Microbiol.,62(12): 4345-4351, 1996) was introduced into theCJ3P::gltA(N241T)-hom(G378E) strain used in Example 7, was prepared.Additionally, a strain, in which only the ilvA(V323A) modification wasintroduced to the CJ3P::hom(G378E), was prepared as the control group.Recombinant vectors for the introduction of modifications were preparedby the method described below.

To prepare a vector for the introduction of ilvA(V323A), first, primers(SEQ ID NO: 55 and 56), in which a recognition site of the restrictionenzyme (XbaI) was inserted into the 5′-end fragment and the 3′-endfragment, respectively, about 600 bp apart either downstream or upstreamfrom the positions of the 966^(st) to the 969^(th) nucleotide sequencesof the ilvA gene, were synthesized using the genomic DNA extracted fromthe WT strain as a template. Additionally, primers (SEQ ID NO: 57 and58) for substituting the nucleotide sequence of the ilvA gene (Table 9).The pDZ-ilvA(V323A) plasmid was prepared in a form where the DNAfragments (600 bp each) located at each of the 5′ and 3′ ends wasconnected to the pDZ vector (Korea Patent No. 2009-0094433). The 5′-endfragment of the ilvA gene was prepared by PCR using the chromosome ofthe WT strain as a template along with primers (SEQ ID NO: 55 and 57).PCR was performed as follows: denaturation at 94° C. for 2 minutes; 30cycles of denaturation at 94° C. for 1 minute, annealing at 56° C. for 1minute, and polymerization at 72° C. for 40 seconds; and polymerizationat 72° C. for 10 minutes.

Likewise, 3′-end fragment of the ilvA gene was prepared by PCR usingprimers (SEQ ID NO: 56 and 58). The amplified DNA fragment was purifiedusing the PCR Purification kit (Quiagen) and used as the insertion DNAfragment for vector preparation. Meanwhile, the pDZ vector, which wastreated with the restriction enzyme XbaI and heat treated at 65° C. for20 minutes, was ligated to the insertion DNA fragment amplified by PCRusing the Infusion Cloning Kit, and the ligated product was transformedinto E. coli DH5α and plated on a solid LB medium containing kanamycin(25 mg/L). The colonies, transformed with a vector in which the targetgene obtained by PCR using primers (SEQ ID NO: 55 and 56) was inserted,were selected and the plasmid was obtained by a conventionally knownplasmid extraction method, and thereby a vector for the introduction ofa modification of nucleotide substitution of ilvA(V323A) on thechromosome (i.e., pDZ-ilvA(V323A)) was prepared.

TABLE 9 Primer Sequence (5′ → 3′) Primer (SEQ ID NO: 55)ACGGATCCCAGACTCCAAAGCAAAAG CG Primer (SEQ ID NO: 56)ACGGATCCAACCAAACTTGCTCACAC TC Primer SEQ ID NO: 57)ACACCACGGCAGAACCAGGTGCAAAG GACA Primer (SEQ ID NO: 58)CTGGTTCTGCCGTGGTGTGCATCATC TCTG

Strains in which a nucleotide modification is introduced in the ilvAgene in the CJ3P::hom(G378E) and CJ3P::gltA(N241T)-hom(G378E) strains bythe pDZ-ilvA(G378E) vector using the method same as in Example 6 (i.e.,CJ3P::hom(G378E)-ilvA(V323A) andCJ3P::gltA(N241T)-hom(G378E)-ilvA(V323A)) were obtained. The obtainedtwo kinds of strains were cultured using the method same as in Example5, and the concentration of isoleucine and GA of the culture broth andsugar consumption rate were analyzed. The results are shown in Table 10below.

TABLE 10 Concentration of isoleucine, sugar consumption rate andcomposition of culture broth Ile GA Sugar Concentration ConcentrationConsumption Strain (g/L) (mg/L) Rate (g/hr) CJ3P::hom(G378E)-ilvA(V323A)0.5 2912 4.92 CJ3P::gltA(241T)-hom(G378E)-ilvA(V323 A) 1.6 2006 5.13

As a result of the analysis of the concentration of threonine, sugarconsumption rate and composition of culture broth, it was confirmed thatthe isoleucine concentration increased at a similar level of sugarconsumption rate while the glutamic acid concentration decreased, in thestrain where the modification of gltA(N241T) was introduced.

From the foregoing, a skilled person in the art to which the presentdisclosure pertains will be able to understand that the presentdisclosure may be embodied in other specific forms without modifying thetechnical concepts or essential characteristics of the presentdisclosure. In this regard, the exemplary embodiments disclosed hereinare only for illustrative purposes and should not be construed aslimiting the scope of the present disclosure. On the contrary, thepresent disclosure is intended to cover not only the exemplaryembodiments but also various alternatives, modifications, equivalents,and other embodiments that may be included within the spirit and scopeof the present disclosure as defined by the appended claims.

The invention claimed is:
 1. A method for producing an L-amino acid,comprising: culturing a microorganism in a medium, wherein themicroorganism is a microorganism of the genus Corynebacterium comprisinga modified polypeptide having citrate synthase activity, wherein the241st amino acid in the amino acid sequence of SEQ ID NO: 1, asparagine,is substituted with another amino acid and wherein the modifiedpolypeptide has attenuated citrate synthase activity compared to theunmodified polypeptide; and recovering an L-amino acid from the culturedmicroorganism or medium.
 2. The method according to claim 1, wherein the241^(st) amino acid in the amino acid sequence of SEQ ID NO: 1,asparagine, is substituted with an amino acid other than lysine.
 3. Themethod according to claim 1, wherein the another amino acid is anaromatic amino acid or a hydrophilic amino acid.
 4. The method accordingto claim 1, wherein the 241^(st) amino acid in the amino acid sequenceof SEQ ID NO: 1, asparagine, is substituted with threonine, serine, ortyrosine.
 5. The method according to claim 1, wherein the microorganismof the genus Corynebacterium is Corynebacterium glutamicum.
 6. Themethod according to claim 1, wherein the L-amino acid is anaspartate-derived L-amino acid.
 7. The method according to claim 1,wherein the L-amino acid is at least one L-amino acid selected from thegroup consisting of lysine, threonine, methionine, homoserine or aderivative thereof, and isoleucine.